The long term goal of our work is to publish studies that identify novel mechanisms that will contribute to our understanding of how in utero polycyclic aromatic hydrocarbon (PAH) exposures contribute to adverse neurotoxicological outcomes. The revised hypothesis of this proposal is that prenatal B(a)P exposure causes behavioral deficits as a result of altered Sp4 gene product and target expression to result in reductions in target gene-driven neural activity during the time when synapses are forming for the first time. Wild type Cytochrome P450 reductase designated Cprlox/lox and the null genotype will be used in these studies. Such null mice no longer possess the ability to produce B(a)P metabolites. This hypothesis will be tested by using both in vivo and ex vivo mouse models. Aim 1 will determine whether in utero B(a)P exposure leads to behavioral deficits and alteration of Sp4 gene product and target expression in Cprlox/lox offspring as compared to brain-Cpr-null offspring. We propose to use Spatial discrimination reversals and Repeated acquisition of behavioral chains tests that are known to be sensitive to developmental neurotoxicant exposure. Quantitative markers of exposure and disposition will include the generation of metabolites and neuroprostanes in offspring cortex. Predicted outcomes: Prenatal B(a)P exposure will result in behavioral deficit phenotypes in B(a)P-exposed Cprlox/lox offspring and brain-Cpr-null offspring. Aim 2 will determine the molecular mechanism of AhR-dependent regulation of Sp4 gene expression in primary neuronal cultures derived from Cprlox/lox offspring. Transcriptional read out of GFP fluorescence from pLenti6.2-GW/EmGFP-NR2A deletion reporter constructs will be performed. Transduction of lentiviral constructs in Cpr derived primary neuronal cultures in the presence of B(a)P will determine the requirement for XREs and GC-box binding in exposure induced modulation of Sp4 and target gene expression. Predicted outcome;B(a)P effects on Sp4 target gene expression are a function of XREs in the 5'region of Sp4 transcription factor and GC-box binding sites in target gene promoters. Aim 3 will determine the causal relationships between B(a)P-induced: 1) modulation of behavioral deficit phenotypes with 2) modulation of Sp4 gene product and target gene expression with 3) reductions in NMDA NR2A-driven neural function. NMDA-driven neuronal activity will be measured by 1) whole-cell patch clamp analysis from ex vivo primary cultures and 2) ex vivo recordings from cortical slice preparations derived from control and B(a)P-exposed Cprlox/lox offspring. Predicted outcomes;Prenatal B(a)P exposure negatively modulates NR2A-driven currents in offspring. Administration of selective NR2A antagonist in slices derived from exposed Cprlox/lox offspring will demonstrate negative attenuation of these affected excitatory post synaptic potentials. PUBLIC HEALTH RELEVANCE: This revised proposal represents an effort towards understanding how in utero exposures during critical windows of susceptibility produce negative effects on neural function and behavior in offspring progeny. Reconstituting NR2A-driven currents that are suppressed during critical periods of development may be possible as a targeted NR2A agonist therapy at birth-as a means to preserve the development of normal circuitry and thus normal behavior. Translating these new concepts as a result of animal studies offers the promise of advancing our ability to establish gene x environment connections between exposure-induced disease states due to disturbances in temporal expression patterns during critical developmental periods.